1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to methods and compositions for modifying plant flavonoid metabolism.
2. Description of the Related Art
Isoflavonoids represent a class of bioactive plant natural products with important implications for plant, animal and human health (Dixon and Steele, 1999). Over 850 isoflavonoid aglycones have been identified and much of this molecular diversity is generated through the biosynthetic modification of core isoflavonoid chemical scaffolds (Harborne, 1994). The majority of naturally occurring isoflavonoids are O-methylated at one or more positions, and over 80% of the reported isoflavonoids in the forage legume alfalfa (Medicago sativa) contain at least one methoxyl group (Bisby et al., 1994). Methylation alters the chemical reactivity and biosynthetic fate of hydroxyl groups, modulates solubility and intracellular localization (Ibrahim et al., 1987), and re-directs biosynthetic intermediates down specific branches of complex metabolic grids (Maxwell et al., 1993).
Enzymatic O-methylation is catalyzed by O-methyltransferases (OMTs), which catalyze transfer of a methyl group from S-adenosyl-L-methionine (SAM) to a hydroxyl moiety of an acceptor molecule. Two distinct groups of small molecule OMTs can be distinguished from plants (Joshi and Chiang, 1998). Group I OMTs are 38-43 kDa proteins which do not require a metal ion for activity, and which methylate a variety of acceptors including phenylpropanoids, flavonoids, alkaloids, and coumarins (Noel et al., 2003). Group II OMTs are of lower MW (23-27 kDa), and are Mg2+-dependent enzymes represented by caffeoyl-CoA 3-OMT. Substrate specificities of OMTs can not be accurately predicted on the basis of sequence similarity alone (Schroder et al., 2002), and in some cases a single amino acid change can alter substrate specificity (Frick and Kutchan, 1999; Gang et al., 2002). Elucidation of the crystal structures of caffeoyl CoA 3-OMT and three different group I OMTs from alfalfa (Zubieta et al., 2001; Zubieta et al., 2002; Ferrer et al., 2005) has now made it possible to explore the structural basis of OMT substrate specificity by homology-based modeling (Hoffmann et al., 2001; Gang et al., 2002; Komblatt et al., 2004; Yang et al., 2004).
Isoflavonoid OMTs catalyze the biosynthesis of natural chemicals that are important for disease resistance in legumes. 6a-Hydroxymaackiain 3-OMT in pea (Pisum sativum) (PsHMM) methylates the relatively nontoxic isoflavonoid-derived compound 6a-hydroxymaackiain to produce the potent antimicrobial chemical pisatin, thereby constituting an essential step in phytoalexin biosynthesis (VanEtten et al., 1982; Preisig et al., 1989).
In addition to PsHMM, two other isoflavonoid O-methyltransferases have been characterized at the molecular level. Medicago sativa isoflavone 7-OMT (MsI7OMT) catalyzes A-ring 7-O-methylation of isoflavones such as daidzein in vitro (FIG. 1), and has been studied both biochemically and structurally (He and Dixon, 1996; He et al., 1998; Zubieta et al., 2001; U.S. Patent Publication 20050150010). Although 7-O-methylated isoflavones such as isoformononetin (7-O-methyl-daidzein) are uncommon in legumes, the induction of I7OMT transcripts and observable enzymatic activity after elicitation or fungal infection in alfalfa suggested a role for this enzyme in the phytoalexin response (Edwards and Dixon, 1991; Akashi et al., 2000; He and Dixon, 2000). Paradoxically, over-expression of MsI7OMT in alfalfa did not produce isoformononetin, but led to greater accumulation of the 4′-O-methylated isoflavonoids formononetin and medicarpin in elicited leaves, and enhanced resistance to the fungal leaf pathogen Phoma medicaginis (He and Dixon, 2000). I7OMT activity is not restricted to alfalfa, and the enzyme has been recently cloned from licorice (Glycyrrhiza echinata) (Akashi et al., 2003). In addition to their role in plant defense responses, isoflavones may also demonstrate medical or nutraceutical uses.
The most common site of methylation of isoflavonoids is at the 4′-position of the B-ring (isoflavone numbering), but identification of an IOMT with 4′-specificity eluded efforts until recently (Wengenmayer et al., 1974; Edwards and Dixon, 1991; He and Dixon, 1996; Akashi et al., 2003). It now appears that 4′-O-methylation in isoflavonoid biosynthesis occurs at the level of 2-hydroxyisoflavanone, the direct product of the “isoflavone synthase” (IFS) that constitutes the entry point into the isoflavonoid pathway (Akashi et al., 2000). 2,7,4′-Trihydroxyisoflavanone 4′-OMT (HI4′OMT; e.g. GenBank accession AB091686) catalyzes the methylation of the 4′-position of 2-hydroxyisoflavanone to form 2,7-dihydroxy, 4′-methoxyisoflavanone, which undergoes dehydration to yield the isoflavone formononetin (FIG. 1). Formononetin accumulates in several legumes and is also a precursor in the biosynthesis of medicarpin and related pterocarpanoid phytoalexins. HI4′OMT is closely related to PsHMM at the amino acid sequence level (Asamizu et al., 2000; Akashi et al., 2003). GenBank accession AY942158 describes a M. truncatula isoflavonoid methyltransferase.
Recently, a 4′-OMT from Glycine max (SOMT-2) was reported to methylate daidzein, genistein, and naringenin (Kim et al., 2005). SOMT-2 is 67% identical to MsI7OMT and MtIOMT1 and only 48-53% identical to OMTs within the HI4′OMT clade. However, in contrast to the IOMTs described below, SOMT-2 methylated apigenin and quercetin.
An extensive metabolite profile of M. truncatula roots or root exudates has not been reported. Further evidence of the role of MtIOMTs in isoflavonoid biosynthesis awaits reverse genetic approaches coupled with extensive metabolic profiling to include secreted compounds. It has also been observed that, at least in vitro, some plant small molecule OMTs may form heterodimers with altered substrate preferences and specificities (Frick et al., 2001), although it is unclear whether such a phenomenon can occur in vivo.
While the foregoing studies have provided a further understanding of the biosynthesis of plant secondary metabolites, methods for the efficient modification of most secondary metabolites have been lacking. This has been particularly true in the case of isoflavonoid biosynthesis. There therefore remains a great need in the art for the development of methods and compositions that would increase the efficiency by which isoflavonoid biosynthesis can be modified in plants.